Because of their capability of killing both without prior sensitization and without major histocompatibility requirements, natural killer (NK) cells represent an immune surveillance mechanism independent of classic T cell-mediated immunity . Studies with human and animal subjects have revealed that NK cells are a first-line defense against viruses, bacteria, parasites, and tumors [2-5]. Compromised or absent natural immunity is associated with acute and chronic viral infections such as AIDS, chronic fatigue immune dysfunction syndrome (CFIDS), psychiatric depression, and various immunodeficiency syndromes [6, 7]. Although its nature is much debated, CFIDS is generally agreed to be characterized by debilitating fatigue and by a number of immunologic abnormalities, the most consistent being a significant depression of NK cell activity [8-10]. The present study was undertaken to determine whether there is an association between low levels of NK cell activity and the severity of clinical conditions of patients with clinically defined CFIDS. Our results confirm and extend previous reports that low NK cell cytotoxicity is a pronounced immunologic abnormality found in some patients with CFIDS [8-10].
Materials and Methods
Subjects. Twenty patients (14 females and six males) with clinically defined CFIDS were part of a group of patients in Flint, Michigan, under the care of one of the authors (E.J.C.). All patients met the diagnostic criteria for CFIDS established by the Centers for Disease Control and Prevention (Atlanta). . Blood samples were obtained from these patients and from 50 control subjects whom were also in Flint. The ages of the patients and control subjects ranged from 24 to 50 years. The blood specimens were obtained from all subjects by venipuncture and were collected in 15-mL Vacutainer tubes (Becton Dickinson Vacutainer Systems. Rutherford, NJ) containing preservative-free heparin. The blood samples were drawn at 3:00 P.M. in Michigan and shipped overnight at ambient temperature to reach Specialty Laboratories in Santa Monica, California, by 9:00 the following morning. In previous experiments, we established that blood samples are still suitable for NK cell function tests 18-24 hours after collection. However, there is a 10%-20% decrease in NK cell activity in such samples as compared with that in freshly drawn blood.
Cytotoxicity assay. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweeden) centrifugation and used as effector cells. NK cell activity was assayed by the measurement of the release of 51Cr in a standard cytotoxicity assay, as previously described . All experiments were performed in triplicate in round-bottomed microtiter plates in RPMI-1640 medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. Assays were performed at four effector-to-target ratios (50:1, 25:1, 12.5:1, and 6.25:1) with use of 2 x 104 target cells/well. K562, an NK cell-sensitive chronic myelogenous leukemia cell line, was used as a source of target cells. The percentage of lysis was converted to lytic units (LU) according to the method of Bryant et al. .
Categorization of patients with CFIDS. Independently of the double-blind measurement of NK cell activity, the 20 patients with CFIDS were globally stratified by one physician into three groups according to duration and severity of their previous physical condition (table 1), as outlined below. On the basis of a group scoring system of 1-10 points, with 10 being the most severe clinical status, the patients were classified as follows: group A, >7 points; group B, 5-7 points; and group C, <5 points.
On a scale of 1-10, the physical condition of each patient was rated according to the duration of the following features: exhaustion/fatigue, postexertional weakness, arthralgia, myalgia, muscle weakness, twitching, depression, short-term memory loss, neuroirritability, sleep disorders, frequent headaches, fever, chills, nausea/vomiting, abdominal pain, sore throat, lymph node pain, and lymph node enlargement.
Clinical findings were obtained by a standard physician's examination and globally rated on a scale of 1-10 in regard to the following parameters: temperature; heart rate; respiration rate; findings on examination of lymph nodes (palpable, tender), throat, chest, abdomen (tender), and muscles (tender, lumps); neurological findings; and results of electrocardiography, electroencephalography, and electromyography.
The subjective severity (based on the duration estimated by each patient) of medically documented and other illnesses or conditions that had occurred in the previous 5 years was globally rated by the physician on a scale of 1-10. The following conditions were considered: viral infections, psychological trauma, complications with pregnancy, and other illnesses enumerated as part of the medical history.
Personal details (occupation, gender, and race) were also recorded: most of the patients (14 of 20) were Caucasian females who were young adults.
Statistical Analysis. Statistical comparisons were made with the Student's t-test.
Results and Discussion
The reference range of NK cell activity established for the 50 healthy controls was 20-250 LU. For 25 (25%), measurements were 20-50 LU; for 16 (32%), 51-100 LU; for 3 (6%), 101-130 LU; and for 6 (12%), >150 LU (figure 1). In contrast to that in 18% of the controls, in none of the 20 patients with CFIDS was NK cell activity > 100 LU. Those stratified as least severely affected (group C; 10 patients [50%]) had NK cell activity of 61.0 ± 21.7 LU; those with conditions of intermediate severity (group B: 7 patients [35%]) had measures of 18.3 ± 7.3 LU; and the most severely affected (group A: 3 patients [15%]) had measures of 8.0 ± 5.3 LU (figure 1). The NK cell activity of all three groups (A + B + C) was significantly lower than that of the control group (P < .05). Of the three CFIDS groups, only group B (intermediate severity) had NK cell activity that was significantly lower than that of the control group (P <.05). The NK cell activity of group C (least severity) was not significantly different from that of the control group (P = 1.0); this group compromised patients with complaints of the shortest duration and with the least severe affliction according to the global assessment. Although the mean LU of activity in group A (most severity) was lower than that in the control group, the difference between the means was not statistically significant (P = 0.1), a finding that possibly is a reflection of the small sample size of group A.
Additional blind, controlled studies of NK cell activity in larger groups of patients with CFIDS and in patients with clinically similar problems are needed to validate the stratification of patients with CFIDS into distinct groups. OF course, there almost certainly was substantial overlapping in the complaints of patients in group C and of the control group subjects, perhaps reflecting the fact that those patients' complaintss were of shorter duration than those of group A and B patients. Nevertheless, the preliminary data suggests that a relatively crude, admittedly less-than-objective global stratification of the patients with CFIDS into distinct groups according to the severity or duration of past and current complaints and physical abnormalities might allow identification of laboratory abnormalities that are possibly associated with severity.
In conclusion, whether the relatively low level of NK cell activities observed in some patients with CFIDS are a cause or effect of CFIDS is presently unknown. We are currently studying a larger group of patients with CFIDS who have been stratified by a similar system with regard to NK cell activity, the presence of antibodies to the early antigen of human herpes virus 6, and the presence of other possibly relevant analytes. However, the fact that NK cell activity decreases with the increased severity and duration of certain clinical variables suggests that measurement of NK cell function could be useful for stratification of patients and possibly for monitoring therapy for and/or the progression of CFIDS.
The authors are very grateful to Diane Roberts, R.N., for her assistance in providing the patients with CFIDS, and to Shirley C. Chang for technical assistance.
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